1. Field of the Invention
This invention relates to a method for determining .gamma.-glutamyltransferase activity as well as to a kit containing a novel substrate solution for use therein.
2. Description of the Prior Art
.gamma.-glutamyltransferase [(.gamma.-glutamyl)-peptide:amino acid .gamma.-glutamyltransferase (EC 2.3.2.2)], discovered in 1950 by Hanes et al. (1, 2), catalyzes the transfer of the .gamma.-glutamyl group from a .gamma.-glutamyl peptide to an amino acid or another peptide.
Although .gamma.-glutamyltransferase is widely distributed in human organs, an increase in its activity in serum is almost specifically an indicator of diseases of the hepatobiliary track or of hepatic involvement in the primary elements (3-7).
For determination of .gamma.-glutamyltransferase activity, L-.gamma.-glutamyl-p-nitroanilide (8) is at present almost invariably used as a substrate, because it allows a direct reaction rate measurement without deproteinization or any chemical treatment of the cleavage product, p-nitroanaline. Recently, a more soluble derivative of L-.gamma.-glutamyl-p-nitroanilide, namely, L-.gamma.-glutamyl-3-carboxy-p-nitroanilide has been described as an alternative substrate in the .gamma.-glutamyltransferase assay (9).
Prior art solutions of both L-.gamma.-glutamyl-3-carboxy-p-nitroanilide and L-.gamma.-glutamyl-p-nitroanilide suffer from stability and solubility problems in their presently employed aqueous environments. For example, the relatively short shelf-life of aqueous solutions of both L-.gamma.-glutamyl-3-carboxy-p-nitroanilide and L-.gamma.-glutamyl-p-nitroanilide have necessitated the freeze-drying thereof to prolong the shelf-life of these products. To reconstitute the freeze-dried product requires one to place it in water and heat the resulting mixture to up to about 50.degree. C. This reconstitution step is time consuming, causes greater variations in the resulting reagent, as well as a loss of substrate. In addition, the freeze-drying procedure itself creates larger lot-to-lot variations.
Accordingly, it would be very advantageous to develop a novel substrate solution for use in the assay of .gamma.-glutamyltransferase wherein such substrate solution comprises a substrate dissolved in a matrix such that the resulting substrate solution possesses increased stability and overcomes the solubility problems present in prior art.